HIV-I is a member of the viral family, Retroviridae. These pleomorphic, enveloped lentiviruses range from 90 to 120 nm in diameter, with a genome consisting of two single stranded RNA molecules. HIV-I particles contain a reverse transcriptase enzyme which converts the RNA genome into a single double-stranded DNA molecule following cell penetration. HIV-I DNA may integrate into the genome of the infected cell and remain latent for several years in a carrier person. It causes persistent infections in CD4+ cells of a person's immune and central nervous systems. Progression from an asymptomatic state acquired immune deficiency syndrome (AIDS) is associated with the depletion of the helper T cell population, and a general breakdown of both cellular and humoral immune functions. AIDS represents the severe manifestation of a viral infection that produces a broad range of clinical effects, and results in death.
The diagnosis of HIV-I infection is accomplished by detecting antibodies to the virus, direct detection of viral antigens, culture isolation from patient specimens, or direct detection of nucleic acids from free virus or viral-infected cells. Although serological assays provide a rapid, inexpensive and relatively sensitive screen for previous exposure to HIV-I, reactive samples must be confirmed as positive with supplemental tests such as immunofluorescent, Western blot or radioimmunoprecipitation assays.
The direct detection of HIV-I is more desirable than indirect serological assays because maternal antibodies persist for up to 15 months in newborns, and direct detection can distinguish between maternal and newborn infection. Moreover, direct detection allows the identification of infected individuals in which the antibodies have not yet formed. Direct viral detection can also be used as a confirmatory test for serological indeterminates. Due to the extremely low concentrations (copy number) of HIV-I present in early stages of infection, conventional direct detection methods perform poorly without first culture amplifying the specimen. Such cultures for HIV-I are technically demanding, time consuming and expensive.
Technology to detect minute quantities of nucleic acids (including HIV-I DNA) has advanced rapidly over the last ten years including the development of highly sophisticated hybridization assays using probes in amplification techniques such as PCR. Researchers have readily recognized the value of such technology to detect diseases and genetic features in human or animal test specimens. The use of probes and primers in such technology is based upon the concept of complementarity, that is the bonding of two strands of a nucleic acid by hydrogen bonds between complementary nucleotides (also known as nucleotide pairs).
PCR is a significant advance in the art to allow detection of very small concentrations of a targeted nucleic acid. The details of PCR are described, for example, in U.S. Pat. No. 4,683,195 (Mullis et al), U.S. Pat. No. 4,683,202 (Mullis), and U.S. Pat. No. 4,965,188 (Mullis et al) and by Mullis et al, Methods of Enzymology, 155, pp. 335-350 (1987), although there is a rapidly expanding volume of literature in this field. Without going into extensive detail, PCR involves hybridizing primers to the strands of a targeted nucleic acid (considered "templates") in the presence of a polymerization agent (such as a DNA polymerase) and deoxyribonucleoside triphosphates under the appropriate conditions. The result is the formation of primer extension products along the templates, the products having added thereto nucleotides which are complementary to the templates.
Once the primer extension products are denatured, one copy of the templates has been prepared, and the cycle of priming, extending and denaturation can be carried out as many times as desired to provide an exponential increase in the amount of nucleic acid which has the same sequence as the target nucleic acid. In effect, the target nucleic acid is duplicated (or "amplified") many times so that it is more easily detected. Despite the broad and rapid use of PCR in a variety of biological and diagnostic fields, there are still practical limitations which must be overcome to achieve the optimum success of the technology.
It is well known that PCR is susceptible to a "carry-over" problem whereby amplified nucleic acids from one reaction may be inadvertently carried over into subsequent reactions using "fresh" PCR reaction mixtures, and thereby causing "false" positives when testing later specimens.
One approach to this problem is to completely contain the reagents for each PCR procedure so no reagents or by-products can be carried over into later reactions. Specially designed test packs or test devices have been designed to contain PCR procedures for this reason. Such test packs are described, for example, in recently allowed U.S. Ser. No. 07/962,159 [filed Oct. 15, 1992 by Schnipelsky et al as a continuation of U.S. Ser. No. 07/673,053 (filed Mar. 21, 1991, now abandoned) which in turn is a CIP of U.S. Ser. No. 07/339,923 (filed Apr. 17, 1989, now abandoned) which in turn is a CIP of U.S. Ser. No. 07/306,735 (filed Feb. 3, 1989, now abandoned)]. These test devices are preferably, but not necessarily, used in PCR in combination with automatic PCR processing equipment such as that described in U.S. Pat. No. 5,089,660 (Devaney Jr.) and in U.S. Pat. No. 5,089,233 (Devaney Jr. et al). This equipment is characterized by its capability to simultaneously process several test specimens in separate test devices.
More preferably, it would be desirable to detect a multiplicity of target nucleic acids (or a multiplicity of nucleic acid sequences in the same nucleic acid) in a single test device. This is referred to herein as "multiplexing".
In one embodiment of PCR, a specific set of primers and a capture probe (a total of three oligonucleotides) are needed for each target nucleic acid which is to be amplified and detected. Thus, the three oligonucleotides are complementary and specific to that target nucleic acid. For example, in multiplexing, if three target nucleic acids are to be amplified and detected, three sets of primers and probes are needed, one set specific for each target nucleic acid. Normally, detection of the multiple nucleic acids requires a multiplicity of test devices, and perhaps different sets of PCR conditions (that is, temperature and time conditions) to obtain efficient amplification of each target nucleic acid.
It would be desirable, however, to amplify and detect a plurality of target nucleic acids simultaneously in the same test device, using "universal" processing equipment and conditions. This cannot be done by merely selecting any set of primers and probes specific for each target nucleic acid from conventional sources. Where processing equipment is used to process several test devices simultaneously, or a single test device is designed for multiplexing, the equipment must be somehow adapted to provide optimum heating and cooling times and temperatures for each set of primers and probes, since they will all likely have individual optimum amplification conditions (for example, denaturaton temperatures). To adapt the equipment to randomly selected primers and probes in multiplexing would be a very expensive and cumbersome solution to the problem. Yet there is a great need for efficient, relatively inexpensive and rapid multiplexing to detect multiple nucleic acid sequences of a retroviral DNA, or one or more nucleic acid sequences of a retroviral DNA and one or more nucleic acid sequences of other target nucleic acids.